Project Summary In the last two decades the eukaryotic cell cycle has gone from an enigma to a well articulated branch of cell biology. Two major gaps are apparent: integration of the cell cycle into aspects of cell physiology like growth and differentiation and establishing mechanistic understandings, especially around the complex processes of ubiquitylation and protein degradation. We address one aspect of the former and more broadly, the latter. Although ubiquitylation and phosphorylation are the major known regulatory mechanisms in the cell cycle, other modifications such as sumoylation and neddylation have been shown to have critical and specific roles. Using a recently developed protein array technology, we show that modification by FAT10, a ubiquitin-like protein that may serve as a degradation signal, is broadly regulated at the Metaphase/G1 transition. Building on our recent demonstration of the functional importance of Fat10ylation we plan to validate target proteins and use them to study the biochemical steps of FAT10ylation. We plan to examine whether FAT10 has a broad role in mitosis and to examine the biological role of particular substrates, focusing on those involved in metaphase arrest and apoptosis. The second project confronts the difficulty in resolving heterogeneous intermediates in the ubiquitin pathway and of identifying the logic of the pathway, key points of regulation and specificity. In particular the lack of mechanistic and dynamical understanding has stymied efforts to identify features that determine the timing and rate of degradation. Single molecule enzymatic studies have proven to be capable of resolving unappreciated mechanistic features of well studied reactions, but have not previously been used to analyze entire pathways. We have begun to develop single-molecule methods to probe ubiquitylation mediated by the anaphase promoting complex, including deubiquitinating enzymes, E2's and the proteasome. We describe initial experiments that have already shed light on previously impenetrable features. By combining these approaches with the production of substrates with chemically specified ubiquitin configurations we hope to learn how ubiquitin configuration controls the timing, specificity, and rate of degradation. Expanding these single molecule studies to the proteasome may reveal additional features of specificity. These studies offer real promise of predictive models for interpreting sequence features that serve as a code for protein degradation and may inform new pharmacological development.